Purification of factor xiii

Abstract

Methods for purifying factor XIII from a biological fluid are provided. The methods comprise precipitation of factor XIII by adjusting the pH of the biological fluid to 5.5 to 6.5 and recovering the precipitated factor XIII.

We claim: 
     
       1. A method for purifying factor XIII from a biological fluid, comprising: precipitating the factor XIII by adjusting the pH of the biological fluid to 5.5 to 6.5; and   recovering the precipitated factor XIII.   
     
     
       2. The method of claim 1 wherein the pH of the fluid is adjusted by the use of a low ionic strength, polyamine or phosphate buffer. 
     
     
       3. The method of claim 2 wherein the low ionic strength polyamine is selected from the group consisting of piperazine and derivatives thereof. 
     
     
       4. The method of claim 1 wherein the precipitating step comprises dialyzing the biological fluid in a buffer comprising 10-100 mM piperazine, pH 6.0. 
     
     
       5. The method of claim 1, further comprising washing the recovered, precipitated factor XIII. 
     
     
       6. A method for purifying factor XIII from a biological fluid, comprising: fractioning the biological fluid by anion exchange chromatography to produce an enriched fraction;   precipitating the factor XIII by adjusting the pH of the enriched fraction to 5.5 to 6.5; and   recovering the precipitated factor XIII.   
     
     
       7. The method of claim 6, comprising the additional steps of: dissolving the recovered, precipitated factor XIII to produce a solution;   fractionating the solution by chromatography to produce a second enriched fraction; and   recovering the second enriched fraction.   
     
     
       8. The method of claim 6 wherein the pH of the enriched fraction is adjusted by the use of a low ionic strength, polyamine or phosphate buffer. 
     
     
       9. The method of claim 8 wherein the low ionic strength polyamine is selected from the group consisting of piperazine and derivatives thereof. 
     
     
       10. The method of claim 6 wherein the precipitating step comprises dialyzing the enriched fraction in a buffer comprising 10-100 mM piperazine, pH 6.0. 
     
     
       11. The method of claim 6 wherein the biological fluid is a cleared cell lysate. 
     
     
       12. The method of claim 11 wherein the cleared cell lysate is a yeast cell lysate. 
     
     
       13. The method of claim 6 wherein the biological fluid is prepared from a cell lysate by: removing particulate material from a cell lysate to produce a cleared lysate;   adding a precipitating agent to the cleared lysate to produce a precipitate; and   resuspending the precipitate in a suitable buffer to form a biological fluid.   
     
     
       14. The method of claim 13 wherein the step of adding a precipitating agent comprises adding ammonium sulfate to between about 30% to 40% of saturation, or adding polyethylene glycol to a concentration of between about 6% to 17% by weight. 
     
     
       15. The method of claim 6 wherein the fractionating step comprises chromatography on DEAE anion exchange media. 
     
     
       16. The method of claim 7 wherein the step of fractionating the solution by chromatography comprises filtration of the solution on gel filtration chromatography media. 
     
     
       17. A method for purifying factor XIII from a biological fluid, comprising: adding to a biological fluid ammonium sulfate to about 30% to about 40% of saturation to produce a precipitate;   resuspending the precipitate in a low ionic strength, mildly alkaline buffer;   fractioning the resuspended precipitate by anion exchange chromatography to produce an enriched fraction;   precipitating the factor XIII by adjusting the pH of the enriched fraction to 5.5 to 6.5 with piperazine buffer;   recovering the precipitated factor XIII;   dissolving the precipitated factor XIII to produce a solution;   fractioning the solution by size exclusion chromatography to produce a second enriched fraction; and   recovering the second enriched fraction.   
     
     
       18. The method of claim 17 wherein the biological fluid is a cleared cell lysate. 
     
     
       19. The method of claim 18 wherein the cleared cell lysate is a yeast cell lysate.